Identification of a hypothetical protein from Podospora anserina as a nitroalkane oxidase.

Abstract

The flavoprotein nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of primary and secondary nitroalkanes to their respective aldehydes and ketones. Structurally, the enzyme is a member of the acyl-CoA dehydrogenase superfamily. To date no enzymes other than that from F. oxysporum have been annotated as NAOs. To identify additional potential NAOs, the available database was searched for enzymes in which the active site residues Asp402, Arg409, and Ser276 were conserved. Of the several fungal enzymes identified in this fashion, PODANSg2158 from Podospora anserina was selected for expression and characterization. The recombinant enzyme is a flavoprotein with activity on nitroalkanes comparable to the F. oxysporum NAO, although the substrate specificity is somewhat different. Asp399, Arg406, and Ser273 in PODANSg2158 correspond to the active site triad in F. oxysporum NAO. The k(cat)/K(M)-pH profile with nitroethane shows a pK(a) of 5.9 that is assigned to Asp399 as the active site base. Mutation of Asp399 to asparagine decreases the k(cat)/K(M) value for nitroethane over 2 orders of magnitude. The R406K and S373A mutations decrease this kinetic parameter by 64- and 3-fold, respectively. The structure of PODANSg2158 has been determined at a resolution of 2.0 A, confirming its identification as an NAO.

The PODANSg2158 active site superimposed on σA-weighted electron density with coefficients 2mFo-DFc contoured at 1.2σ. The FAD isoalloxazine ring, the catalytic triad consisting of residues S273, D399, and R406, and a chain of hydrogen-bonded water molecules running from the active site to the bulk solvent are shown.

Superposition of subunit A of the F. oxysporum NAO structure [PDB code 2C12 (), pink] and subunit A of the PODANSg2158 structure (cyan). The respective flavins are shown in stick representation with the same color scheme.

Overlay of the active sites from PODANSg2158 (carbon atoms colored in pink) and F. oxysporum NAO (carbon atoms colored in cyan). The active site of PODANSg2158 was overlaid with that of F. oxysporum enzyme (PDB code 2C12) () by superimposing the 4 carbons on the central ring in the FADs.

Active site entrances in F. oxysporum nitroalkane oxidase [PDB code 2C12 (), top] and P. anserina PODANSg2158 (bottom). The placement of the 1-nitrooctane (yellow in both panels) is based on the superposition of the backbone atoms of these structures with the backbone atoms of PDB code 2D9E ().

The flavoenzyme nitroalkane oxidase (NAO) from the soil fungus Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the corresponding aldehydes or ketones with the release of nitrite and the consumption of molecular oxygen to yield hydrogen peroxide () (, ). NAO is unusual, since it catalyzes substrate oxidation by removing a substrate proton to form a carbanion intermediate () (), whereas flavoproteins that oxidize carbon-nitrogen and carbon-oxygen bonds typically catalyze cleavage of the substrate carbon-hydrogen bond by removal of a hydride rather than a proton (–). The NAO reaction is initiated by abstraction of the α-proton from the neutral form of the nitroalkane by Asp402, the active site base (), creating a nucleophilic nitroalkane anion (). The anion then attacks the N5 position of FAD to form an adduct; this eliminates nitrite to generate a cationic, electrophilic flavin imine that can be attacked by hydroxide (–). Release of the aldehyde or ketone product forms reduced FAD (). In the more typical oxidative half-reaction, the reduced FAD is oxidized by molecular oxygen to form hydrogen peroxide (). Release of products from the oxidized enzyme limits turnover with primary nitroalkanes, the best substrates (). With the slow substrate nitroethane (), formation of the substrate anion is rate-limiting for the reductive half-reaction ().

The flavoenzyme nitroalkane oxidase (NAO) from the soil fungus Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the corresponding aldehydes or ketones with the release of nitrite and the consumption of molecular oxygen to yield hydrogen peroxide () (, ). NAO is unusual, since it catalyzes substrate oxidation by removing a substrate proton to form a carbanion intermediate () (), whereas flavoproteins that oxidize carbon-nitrogen and carbon-oxygen bonds typically catalyze cleavage of the substrate carbon-hydrogen bond by removal of a hydride rather than a proton (–). The NAO reaction is initiated by abstraction of the α-proton from the neutral form of the nitroalkane by Asp402, the active site base (), creating a nucleophilic nitroalkane anion (). The anion then attacks the N5 position of FAD to form an adduct; this eliminates nitrite to generate a cationic, electrophilic flavin imine that can be attacked by hydroxide (–). Release of the aldehyde or ketone product forms reduced FAD (). In the more typical oxidative half-reaction, the reduced FAD is oxidized by molecular oxygen to form hydrogen peroxide (). Release of products from the oxidized enzyme limits turnover with primary nitroalkanes, the best substrates (). With the slow substrate nitroethane (), formation of the substrate anion is rate-limiting for the reductive half-reaction ().

Previous steady-state kinetic analyses of F. oxysporum NAO have established the steady-state kinetic mechanism as a modified ping-pong mechanism typical of flavoprotein oxidases () (, , ). gives the appropriate rate equation for such a mechanism. The appropriateness of rather than the more complex for a sequential mechanism can readily be determined using the fixed ratio method (). In this approach initial rates are measured at different concentrations of the two substrates, keeping the ratio of their concentrations constant. Curvature in a double reciprocal plot of the resulting data suggests a sequential mechanism, since there will be a term containing the square of the concentration of one substrate in the rate equation (, a = [O2]/[NE]). On the other hand, a linear plot will arise if there is no term in the rate equation containing the concentration of both substrates (). For PODANSg2158 a double reciprocal plot of initial rate vs. nitroethane concentration is linear (), establishing as appropriate and yielding a kcat value of 15 s−1. For such a mechanism, the kcat/KM values of the nitroalkane and oxygen are independent of the concentration of the other substrate. The kcat/KM values for oxygen and nitroethane were determined in separate analyses by varying each at a fixed concentration of the other. These values are given in . The KO2 value for PODANSg2158 is higher than that for the F. oxysporum NAO (), but within the range found for a number of other flavoprotein oxidases (–).